Methods and compositions for improved fertilization and embryonic survival

ABSTRACT

Single nucleotide polymorphic sites at positions 19069 and 25402 of the bovine STAT3 gene are associated with improved fertilization rate and/or improved embryo survival rate. The interactions between these two polymorphisms, and between them and the bovine STAT1 gene and fertilization and early embryonic survival rates were also disclosed. The interactions between STAT3 SNPs, and between STAT1 and STAT3 SNP19069 were highly significant for embryonic survival rate. Also disclosed are nucleic acid molecules, kits, methods of genotyping and marker assisted bovine breeding methods.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority of U.S. App. Ser. No. 61/242,390, filed Sep. 15, 2009, the contents of which are incorporated herein by reference.

This invention was made with government support under 09-CRHF-0-6055 awarded by the USDA/CSREES. The government has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to a method of genetic testing for improved fertilization rate and embryonic survival rate in animals, especially dairy cattle.

BACKGROUND OF THE INVENTION

Dairy cows are significant investments for dairy farmers, yet infertility is a major cause of dairy cow culling and economic loss. Enormous efforts, such as animal breeding and artificial insemination, have been and continue to be invested in ensuring improved breeding programs. The decline in reproductive performance in high-producing dairy cows is a major concern of farmers worldwide (Royal et al., 2000; Dobson et al., 2008). Major factors contributing to this poor performance in dairy cattle are low fertilization rate and early embryonic loss (Santos et al., 2004; Morris and Diskin, 2008). Although genetics account for about one-third of the decline in pregnancy rate of dairy cows (Shook, 2006), the identification of major genes affecting cow fertility has been challenging, probably due to the low accuracy of fertility data collected in the field and to the low heritability of this trait. The heritability of open days and pregnancy rate is about 0.04 (VanRaden et al., 2004).

Typically, artificial insemination in dairy cattle is successful only 30-35% of the time. The reasons for this are not clear. However, it is understood that both biological and environmental factors affect fertility rate. Some environmental factors such as heat, lack of precipitation, and other factors can cause stress in cattle and can drop the fertility rate to 10-15%. Commercial artificial insemination operations often shut down in July and August due to the drop in fertility caused by the hot, dry weather. It is also known that certain bulls are more fertile than others due to their genetic makeup. Identifying highly fertile bulls, however, is a time-consuming and expensive process. It can take 5-10 years of tracking the attempts of artificial insemination using semen from a bull before it can be certified as a quality bull.

Marker-assisted selection, on the other hand, can lower the high cost and reduce the extended time commitment of progeny testing currently used to improve sires, since young bull progeny could be evaluated immediately after birth or even prior to birth for the presence/absence of the marker, and young bulls that are determined by genetic testing to have undesirable markers would never be progeny tested.

There is thus a need for a method of genetically evaluating the bulls, as well as the cows, e.g., by genetic testing, to enable a quick and accurate evaluation of its fertility as well as the survival rate of embryos conceived therefrom.

Signal transducer and activator of transcription (STAT) proteins are a family of 7 structurally and functionally related proteins: STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B, and STAT6 (Darnell, 1997). The STAT proteins are transcription factors that play important roles in cytokine signaling pathways (Kisseleva et al., 2002). Following their phosphorylation by janus-kinases (JAKs), STATs translocate to the nucleus to regulate transcription of different genes. The JAK/STAT pathway was found to be conserved in vertebrates (Hombría and Brown, 2002). Recent studies have shown that STAT proteins are involved in the fertilization process and in early embryonic development (Maj and Chelmonska-Soyta, 2007). Teglund et al. (1998) showed that disruption of the Stat5 gene leads to infertility in female mice as a result of small-sized or absent corpora lutea. Truchet et al. (2004) reported that Stat1 and Stat3 are expressed in mouse oocytes and preimplanation embryos and concluded that these 2 genes might have functional importance in early embryonic development because of their roles in the cell cycle and apoptosis. Takeda et al. (1997) reported that Stat3-deficient mice die before embryonic day 8.5 and concluded that Stat3 is an essential gene for early embryonic survival and that its deficiency cannot be compensated for by other STAT proteins. Khatib et al. (2008a, 2009) showed that the CC genotype in exon 8 of bovine STAT5A was associated with high fertilization and early embryonic survival rates.

Given that several genes of the JAK/STAT pathway have been found to be associated with fertility traits in cattle, STAT1 and STAT3—also members of this pathway—were chosen as candidate genes for fertilization rate and early embryonic survival in cattle. Previously, the present inventor has disclosed that single nucleotide polymorphisms (SNPs) in the STAT5A gene are associated with both milk production and fertility (U.S. patent application Ser. No. 12/267,076), and a SNP in the coding region of STAT1 gene is associated with increased milk yield, milk fat and protein percentages (U.S. patent application Ser. No. 11/624,053).

Interestingly, after their phosphorylation in the cytoplasm by the JAKs, STAT1 and STAT3 interact with each other by forming a heterodimer complex which translocates to the nucleus and binds specific DNA sequences (Kodama et al., 1997).

In order to overcome these challenges, the present inventor has constructed an in-vitro fertilization (IVF) system which has the advantages of a unified environment and well-isolated components of the embryonic development process. Indeed, using this system, SNPs in several genes and interactions between them have been found to be associated with fertilization and early embryonic survival rates (Khatib et al., 2008a,b; Khatib et al., 2009). There remains, however, a need to determine the single gene effects of STAT1 and STAT3 polymorphisms and their interactions on fertilization and embryonic survival rates.

SUMMARY OF THE INVENTION

The present inventor investigated the effects of the interactions between polymorphisms in the bovine STAT1 and STAT3 genes and fertilization and early embryonic survival rates using an in-vitro fertilization system. Two SNPs, SNP25402 and SNP19069, were identified in the STAT3 gene, and single SNP analysis revealed significant association between SNP25402 and fertilization rate. The interactions between these two STAT3 SNPs and between a previously-identified STAT1 SNP, SNP213, and SNP19069 were highly significant for embryonic survival rate.

Accordingly, the present invention provides an isolated nucleic acid molecule comprising at least one polymorphic site selected from the group consisting of position 19069 and position 25402 of SEQ ID NO: 1 (part of the bovine STAT3 gene), and at least 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 contiguous nucleotides (nt) or bases of SEQ ID NO: 1 adjacent to the polymorphic site, wherein the nucleic acid molecule comprises an adenine at the polymorphic position. It is recognized that SEQ ID NO: 1 is already known, and the nucleic acid molecule therefore does not encompass one that consists of SEQ ID NO: 1.

Preferably, the nucleic acid molecule which comprises at least 15, more preferably at least 20, still more preferably at least 25, contiguous bases of SEQ ID NO: 1 adjacent to the polymorphic site. In one embodiment, the isolated nucleic acid molecule comprises not more than 1,500 nt, preferably not more than 1000 nt, more preferably not more than 900 nt, more preferably not more than 800 nt, more preferably not more than 700 nt, preferably not more than 600 nt, more preferably not more than 500 nt, preferably not more than 400 nt, more preferably not more than 300 nt, more preferably not more than 150 nt., preferably not more than 100 nt., still more preferably not more than 50 nt.

The nucleic acid molecule preferably contains the polymorphic site which is within 4 nucleotides of the center of the nucleic acid molecule. Preferably, the polymorphic site is at the center of the nucleic acid molecule.

In another embodiment, the nucleic acid molecule contains the polymorphic site which is at the 3′-end of the nucleic acid molecule.

In another embodiment, the nucleic acid molecule contains the polymorphic site which is at the 5′-end of the nucleic acid molecule.

The present invention also provides an array of nucleic acid molecules comprising at least two nucleic acid molecules described above.

The present invention further provides a kit comprising a nucleic acid molecule described above, and a suitable container.

Also provided is a method for detecting single nucleotide polymorphism (SNP) in bovine STAT3 gene, wherein the STAT3 gene has a nucleic acid sequence of SEQ ID NO: 1, the method comprising determining the identity of a nucleotide at one or both positions 19069 and 25402, and comparing the identity to the nucleotide identity at a corresponding position of SEQ ID NO: 1.

In another embodiment, the present invention provides a method for genotyping a bovine cell, using the method above. Suitable bovine cell may be an adult cell, an embryo cell, a sperm, an egg, a fertilized egg, or a zygote. The identity of the nucleotide may be determined by sequencing the STAT3 gene, or a relevant fragment thereof, isolated from the cell.

In a further embodiment, the present invention provides a method for testing the fertility of a bull cattle, the method comprising collecting a nucleic acid sample from the cattle, and genotyping said nucleic sample as described above, wherein a bull having a STAT3 gene sequence which comprises an adenine at positions 19069 or 25402, or both is selected for breeding purposes.

Preferably, a bull having a STAT3 gene sequence which is homozygously A at one of the above described polymorphic sites is selected for breeding purposes.

Preferably, a bull having a STAT3 gene sequence which is homozygously A at both of the above described polymorphic sites is selected for breeding purposes.

Further provided is a method for selectively breeding cattle using a multiple ovulation and embryo transfer procedure (MOET), the method comprising superovulating a female animal, collecting eggs from said superovulated female, in vitro fertilizing said eggs using semen from a suitable male animal, implanting said fertilized eggs into one or more other females allowing for an embryo to develop, genotyping the developing embryo, and terminating pregnancy if the developing embryo does not have adenine (A) at position 19069 or 25402. Preferably, pregnancy is terminated if the embryo is not A at positions 19069 and 25402.

In a preferred embodiment, the present invention provides a method for selectively breeding dairy cattle, comprising selecting a bull whose STAT3 gene is hemizygously or homozygously adenine at position 19069 or 25402, and using its semen for fertilizing a female animal. Preferably the bull is homozygous with regard to the above SNP site. More preferably, the female animal is also homozygous at the above SNP site.

In another preferred embodiment, the present invention provides a method for testing the fertility of a cattle, the method comprising collecting a nucleic acid sample from the cattle, and genotyping said nucleic sample as described above, wherein a cattle having a STAT3 SNP and a STAT1 SNP213 (FIG. 2; SEQ ID NO: 2) combination described below is selected for breeding purposes.

In a preferred embodiment, selection of animals for breeding purpose is based on the interactions between the various SNP genotypes. In general, based on the results shown in Table 3 below, a bull or a cow which is homozygously GG at SNP19069 and homozygously AA at SNP25402 (designated as “SNP19069/SNP25402=GG/AA”) should not be selected for breeding purposes, and pregnancies with such a genotype should be terminated. On the other hand, a bull or a cow which is homozygously AA at SNP19069 and homozygously CC at SNP25402 (designated as “SNP19069/SNP25402=AA/CC”) is selected for breeding purposes. A bull or a cow which is homozygously AA at SNP19069 and heterozygously AC at SNP25402 (designated as “SNP19069/SNP25402=AA/AC”) can also be selected for breeding purposes. Likewise, a bull or a cow which is homozygously GG at SNP19069 and homozygously CC at SNP25402 (designated as “SNP19069/SNP25402=GG/CC”) can be selected for breeding purposes; a bull or a cow which is heterozygously AG at SNP19069 and homozygously CC at SNP25402 (designated as “SNP19069/SNP25402=AG/CC”) can also be selected for breeding purposes; a bull or a cow which is heterozygously AG at SNP19069 and homozygously AC at SNP25402 (designated as “SNP19069/SNP25402=AG/AC) can be selected for breeding purposes; a bull or a cow which is homozygously AA both at SNP19069 and SNP25402 (designated as “SNP19069/SNP25402=AA/AA) can be selected for breeding purposes; a bull or a cow which is homozygously GG at SNP19069 and heterozygously AC at SNP25402 (designated as “SNP19069/SNP25402=GG/AC) can be selected for breeding purposes; and a bull or a cow which is heterozygously AG at SNP19069 and heterozygously AC at SNP25402 (designated as “SNP19069/SNP25402=AG/AC) can be selected for breeding purposes. In other words, any one of the following genotypes or STAT3 SNP combinations may be selected for breeding purposes: SNP19069/SNP25402=AA/CC; SNP19069/SNP25402=AA/AC; SNP19069/SNP25402=GG/CC; SNP19069/SNP25402=AG/CC; SNP19069/SNP25402=AG/AA; SNP19069/SNP25402=AA/AA; SNP19069/SNP25402=GG/AC; and SNP19069/SNP25402=AG/AC.

Based on the results shown in Table 4 below, a bull or a cow which is homozygously GG at the SNP19069 and homozygously TT at STAT1 SNP (designated as “SNP19069/STAT1 SNP=GG/TT”) should not be selected for breeding purposes, and pregnancies with such a genotype should be terminated. On the other hand, a bull or a cow which is homozygously AA at SNP19069 and homozygously TT at STAT1 SNP (designated as “SNP19069/STAT1 SNP=AA/TT”) is selected for breeding purposes. A bull or a cow which is heterozygously AG at SNP19069 and homozygously TT at STAT1 SNP (designated as “SNP19069/STAT1 SNP=AG/TT”) can also be selected for breeding purposes. Likewise, a bull or a cow which is homozygously GG at SNP19069 and homozygously CC at STAT1 SNP (designated as “SNP19069/STAT1 SNP=GG/CC”) can be selected for breeding purposes; a bull or a cow which is homozygously AA at SNP19069 and heterozygously CT at STAT1 SNP9 (designated as “SNP19069/STAT1 SNP=AA/CT”) can be selected for breeding purposes; a bull or a cow which is heterozygous AG at SNP19069 and heterozygously CT at STAT1 SNP (designated as “SNP19069/STAT1 SNP=AG/CT”) can be selected for breeding purposes; a bull or a cow which is homozygously AA at SNP19069 and homozygously CC at STAT1 SNP (designated as “SNP19069/STAT1 SNP=AA/CC”) can be selected for breeding purposes; a bull or a cow which is heterozygously AG at SNP19069 and homozygously CC at STAT1 SNP (designated as “SNP19069/STAT1 SNP=AG/CC”) can be selected for breeding purposes; and a bull or a cow which is homozygously GG at SNP19069 and heterozygously CT at STAT1 SNP (designated as “SNP19069/STAT1 SNP=GG/CT”) can be selected for breeding purposes. In other words, any one of the following genotypes or SNP19069/STAT1 SNP combinations may be selected for breeding purposes: SNP19069/STAT1 SNP=AA/TT; SNP19069/STAT1 SNP=AG/TT; SNP19069/STAT1 SNP=GG/CC; SNP19069/STAT1 SNP=AA/CT; SNP19069/STAT1 SNP=AG/CT; SNP19069/STAT1 SNP=AA/CC; SNP19069/STAT1 SNP=AG/CC; and SNP19069/STAT1 SNP=CT/GG.

In a preferred embodiment, a method for genotyping a bovine cell is provided, the method comprising obtaining a nucleic acid sample from said cell and determining the identity of a nucleotide of at least one position selected from the group consisting of position 19069 of the bovine STAT3 gene and position 25402 of the bovine STAT3 gene. Preferably, the identity of the nucleotides at both positions is determined. Preferably, the method further comprises determining the identity of a nucleotide of position 213 of the bovine STAT1 gene.

A method for selectively breeding cattle is further provided, wherein the method comprises testing an animal as described above, and selecting the animal as a breeding parent only if the animal comprises a SNP combination selected from the group consisting of SNP19069/STAT1 SNP=AA/TT; SNP19069/STAT1 SNP=AG/TT; SNP19069/STAT1 SNP=GG/CC; SNP19069/STAT1 SNP=AA/CT; SNP19069/STAT1 SNP=AG/CT; SNP19069/STAT1 SNP=AA/CC; SNP19069/STAT1 SNP=AG/CC; SNP19069/STAT1 SNP=CT/GG; SNP19069/SNP25402=AA/CC; SNP19069/SNP25402=AA/AC; SNP19069/SNP25402=GG/CC; SNP19069/SNP25402=AG/CC; SNP19069/SNP25402=AG/AA; SNP19069/SNP25402=AA/AA; SNP19069/SNP25402=GG/AC; and SNP19069/SNP25402=AG/AC.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a portion of the STAT3 gene sequence (SEQ ID NO: 1) where the two polymorphic sites are shown.

FIG. 2 shows a portion of the STAT1 gene sequence (SEQ ID NO: 2) where the polymorphic site is shown.

DETAILED DESCRIPTION OF THE INVENTION

Two positions of the bovine STAT3 gene are found to be polymorphic. The term “polymorphism” as used herein refers to the occurrence of two or more alternative genomic sequences or alleles between or among different genomes or individuals. “Polymorphic” refers to the condition in which two or more variants of a specific genomic sequence can be found in a population. A “polymorphic site” is the locus at which the variation occurs. Polymorphisms generally have at least two alleles, each occurring at a significant frequency in a selected population. A polymorphic locus may be as small as one base pair. The first identified allelic form is arbitrarily designated as the reference form, and other allelic forms are designated as alternative or variant alleles. The allelic form occurring most frequently in a selected population is sometimes referred to as the wild type form. Diploid organisms may be homozygous or heterozygous for allelic forms. A biallelic polymorphism has two forms, and a triallelic polymorphism has three forms, and so on.

Polymorphisms may provide functional differences in the genetic sequence, through changes in the encoded polypeptide, changes in mRNA stability, binding of transcriptional and translation factors to the DNA or RNA, and the like. Polymorphisms are also used to detect genetic linkage to phenotypic variation.

One type of polymorphism, single nucleotide polymorphisms (SNPs), has gained wide use for the detection of genetic linkage recently. SNPs are generally biallelic systems, that is, there are two alleles that an individual may have for any particular SNP marker. In the instant case, the SNPs are used for determining the genotypes of the STAT3 and STAT1 genes, which are found to have strong correlation to fertilization rate and embryonic survival.

The provided sequences also encompass the complementary sequence corresponding to any of the provided polymorphisms. In order to provide an unambiguous identification of the specific site of a polymorphism, the numbering of the original STAT3 sequence in the GenBank is shown in FIG. 1 and is used throughout this disclosure.

The present invention provides nucleic acid based genetic markers for identifying bovine animals with superior breeding (such as fertility and embryo survival rates) traits. In general, for use as markers, nucleic acid fragments, preferably DNA fragments, may be as short as 7 nucleotides (nt), but may preferably at least 12 nt, 15 nt, usually at least 20 nt, often at least 50 nt. Such small DNA fragments are useful as primers for the polymerase chain reaction (PCR), and probes for hybridization screening, etc.

The term primer refers to a single-stranded oligonucleotide capable of acting as a point of initiation of template-directed DNA synthesis under appropriate conditions (i.e., in the presence of four different nucleoside triphosphates and an agent for polymerization, such as, DNA or RNA polymerase or reverse transcriptase) in an appropriate buffer and at a suitable temperature. The appropriate length of a primer depends on the intended use of the primer but typically ranges from 15 to 30 nucleotides. Short primer molecules generally require cooler temperatures to form sufficiently stable hybrid complexes with the template. A primer need not reflect the exact sequence of the template but must be sufficiently complementary to hybridize with a template. The term primer site, or priming site, refers to the area of the target DNA to which a primer hybridizes. The term primer pair means a set of primers including a 5′ upstream primer that hybridizes with the 5′ end of the DNA sequence to be amplified and a 3′, downstream primer that hybridizes with the complement of the 3′ end of the sequence to be amplified.

The term “probe” or “hybridization probe” denotes a defined nucleic acid segment (or nucleotide analog segment) which can be used to identify by hybridizing to a specific polynucleotide sequence present in samples, said nucleic acid segment comprising a nucleotide sequence complementary of the specific polynucleotide sequence to be identified. “Probes” or “hybridization probes” are nucleic acids capable of binding in a base-specific manner to a complementary strand of nucleic acid.

An objective of the present invention is to determine which embodiment of the polymorphisms a specific sample of DNA has. For example, it is desirable to determine whether the nucleotide at a particular position is A or C. An oligonucleotide probe can be used for such purpose. Preferably, the oligonucleotide probe will have a detectable label, and contains an A at the corresponding position. Experimental conditions can be chosen such that if the sample DNA contains an A, they hybridization signal can be detected because the probe hybridizes to the corresponding complementary DNA strand in the sample, while if the sample DNA contains a G, no hybridization signal is detected.

Similarly, PCR primers and conditions can be devised, whereby the oligonucleotide is used as one of the PCR primers, for analyzing nucleic acids for the presence of a specific sequence. These may be direct amplification of the genomic DNA, or RT-PCR amplification of the mRNA transcript of the STAT3 gene. The use of the polymerase chain reaction is described in Saiki et al. (1985) Science 230:1350-1354. Amplification may be used to determine whether a polymorphism is present, by using a primer that is specific for the polymorphism. Alternatively, various methods are known in the art that utilize oligonucleotide ligation as a means of detecting polymorphisms, for examples see Riley et al (1990) Nucleic Acids Res. 18:2887-2890; and Delahunty et al (1996) Am. J. Hum. Genet. 58:1239-1246. The detection method may also be based on direct DNA sequencing, or hybridization, or a combination thereof. Where large amounts of DNA are available, genomic DNA is used directly. Alternatively, the region of interest is cloned into a suitable vector and grown in sufficient quantity for analysis. The nucleic acid may be amplified by PCR, to provide sufficient amounts for analysis.

Hybridization may be performed in solution, or such hybridization may be performed when either the oligonucleotide probe or the target polynucleotide is covalently or noncovalently affixed to a solid support. Attachment may be mediated, for example, by antibody-antigen interactions, poly-L-Lys, streptavidin or avidin-biotin, salt bridges, hydrophobic interactions, chemical linkages, UV cross-linking baking, etc. Oligonucleotides may be synthesized directly on the solid support or attached to the solid support subsequent to synthesis. Solid-supports suitable for use in detection methods of the invention include substrates made of silicon, glass, plastic, paper and the like, which may be formed, for example, into wells (as in 96-well plates), slides, sheets, membranes, fibers, chips, dishes, and beads. The solid support may be treated, coated or derivatized to facilitate the immobilization of the allele-specific oligonucleotide or target nucleic acid. For screening purposes, hybridization probes of the polymorphic sequences may be used where both forms are present, either in separate reactions, spatially separated on a solid phase matrix, or labeled such that they can be distinguished from each other.

Hybridization may also be performed with nucleic acid arrays and subarrays such as described in WO 95/11995. The arrays would contain a battery of allele-specific oligonucleotides representing each of the polymorphic sites. One or both polymorphic forms may be present in the array, for example the polymorphism of position 25402 may be represented by either, or both, of the listed nucleotides. Usually such an array will include at least 2 different polymorphic sequences, i.e. polymorphisms located at unique positions within the locus, and may include all of the provided polymorphisms. Arrays of interest may further comprise sequences, including polymorphisms, of other genetic sequences, particularly other sequences of interest. The oligonucleotide sequence on the array will usually be at least about 12 nt in length, may be the length of the provided polymorphic sequences, or may extend into the flanking regions to generate fragments of 100 to 200 nt in length. For examples of arrays, see Ramsay (1998) Nat. Biotech. 16:4044; Hacia et al. (1996) Nature Genetics 14:441-447; Lockhart et al. (1996) Nature Biotechnol. 14:1675-1680; and De Risi et al. (1996) Nature Genetics 14:457-460.

The identity of polymorphisms may also be determined using a mismatch detection technique, including but not limited to the RNase protection method using riboprobes (Winter et al., Proc. Natl. Acad. Sci. USA 82:7575, 1985; Meyers et al., Science 230:1242, 1985) and proteins which recognize nucleotide mismatches, such as the E. coli mutS protein (Modrich, P. Ann. Rev. Genet. 25:229-253, 1991). Alternatively, variant alleles can be identified by single strand conformation polymorphism (SSCP) analysis (Orita et al., Genomics 5:874-879, 1989; Humphries et al., in Molecular Diagnosis of Genetic Diseases, R. Elles, ed., pp. 321-340, 1996) or denaturing gradient gel electrophoresis (DGGE) (Wartell et al., Nucl. Acids Res. 18:2699-2706, 1990; Sheffield et al., Proc. Natl. Acad. Sci. USA 86:232-236, 1989).

A polymerase-mediated primer extension method may also be used to identify the polymorphism(s). Several such methods have been described in the patent and scientific literature and include the “Genetic Bit Analysis” method (WO92/15712) and the ligase/polymerase mediated genetic bit analysis (U.S. Pat. No. 5,679,524). Related methods are disclosed in WO91/02087, WO90/09455, WO95/17676, U.S. Pat. Nos. 5,302,509, and 5,945,283. Extended primers containing a polymorphism may be detected by mass spectrometry as described in U.S. Pat. No. 5,605,798. Another primer extension method is allele-specific PCR (Ruao et al., Nucl. Acids Res. 17:8392, 1989; Ruao et al., Nucl. Acids Res. 19, 6877-6882, 1991; WO 93/22456; Turki et al., J. Clin. Invest. 95:1635-1641, 1995). In addition, multiple polymorphic sites may be investigated by simultaneously amplifying multiple regions of the nucleic acid using sets of allele-specific primers as described in Wallace et al. (WO 89/10414).

A detectable label may be included in an amplification reaction. Suitable labels include fluorochromes, e.g. fluorescein isothiocyanate (FITC), rhodamine, Texas Red, phycoerythrin, allophycocyanin, 6-carboxyfluorescein (6-FAM), 2′,7′-dimethoxy-4′,5′-dichloro-6-carboxyfluorescein (JOE), 6-carboxy-X-rhodamine (ROX), 6-carboxy-2′,4′,7′,4,7-hexachlorofluorescein (HEX), 5-carboxyfluorescein (5-FAM) or N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA), radioactive labels, e.g. ³²P, ³⁵S, ³H; etc. The label may be a two stage system, where the amplified DNA is conjugated to biotin, haptens, etc. having a high affinity binding partner, e.g. avidin, specific antibodies, etc., where the binding partner is conjugated to a detectable label. The label may be conjugated to one or both of the primers. Alternatively, the pool of nucleotides used in the amplification is labeled, so as to incorporate the label into the amplification product.

It is readily recognized by those ordinarily skilled in the art that in order to maximize the signal to noise ratio, in probe hybridization detection procedure, the polymorphic site should at the center of the probe fragment used, whereby a mismatch has a maximum effect on destabilizing the hybrid molecule; and in a PCR detection procedure, the polymorphic site should be placed at the very 3′-end of the primer, whereby a mismatch has the maximum effect on preventing a chain elongation reaction by the DNA polymerase. The location of nucleotides in a polynucleotide with respect to the center of the polynucleotide are described herein in the following manner. When a polynucleotide has an odd number of nucleotides, the nucleotide at an equal distance from the 3′ and 5′ ends of the polynucleotide is considered to be “at the center” of the polynucleotide, and any nucleotide immediately adjacent to the nucleotide at the center, or the nucleotide at the center itself is considered to be “within 1 nucleotide of the center.” With an odd number of nucleotides in a polynucleotide any of the five nucleotides positions in the middle of the polynucleotide would be considered to be within 2 nucleotides of the center, and so on. When a polynucleotide has an even number of nucleotides, there would be a bond and not a nucleotide at the center of the polynucleotide. Thus, either of the two central nucleotides would be considered to be “within 1 nucleotide of the center” and any of the four nucleotides in the middle of the polynucleotide would be considered to be “within 2 nucleotides of the center,” and so on.

In some embodiments, a composition contains two or more differently labeled oligonucleotides for simultaneously probing the identity of nucleotides or nucleotide pairs at two or more polymorphic sites. It is also contemplated that primer compositions may contain two or more sets of allele-specific primer pairs to allow simultaneous targeting and amplification of two or more regions containing a polymorphic site.

Alternatively, the relevant portion of the STAT3 gene of the sample of interest may be amplified via PCR and directly sequenced, and the sequence be compared to the wild type sequence shown in FIG. 1. It is readily recognized that, other than those specifically disclosed herein, numerous primers can be devised to achieve the objectives. PCR and sequencing techniques are well known in the art and reagents and equipments are readily available commercially.

DNA markers have several advantages; segregation is easy to measure and is unambiguous, and DNA markers are co-dominant, i.e., heterozygous and homozygous animals can be distinctively identified. Once a marker system is established selection decisions could be made very easily, since DNA markers can be assayed any time after a blood sample can be collected from the individual infant animal, or even earlier by testing embryos in vitro if very early embryos are collected. The use of marker assisted genetic selection will greatly facilitate and speed up cattle breeding problems. For example, a modification of the multiple ovulation and embryo transfer (MOET) procedure can be used with genetic marker technology. Specifically, females are superovulated, eggs are collected, in vitro fertilized using semen from superior males and implanted into other females allowing for use of the superior genetics of the female (as well as the male) without having to wait for her to give birth to one calf at a time. Developing blastomeres at the 4-8 cell stage may be assayed for presence of the marker, and selection decisions made accordingly.

In one embodiment of the invention an assay is provided for detection of presence of a desirable genotype using the markers.

The term “genotype” as used herein refers to the identity of the alleles present in an individual or a sample. In the context of the present invention a genotype preferably refers to the description of the polymorphic alleles present in an individual or a sample. The term “genotyping” a sample or an individual for a polymorphic marker refers to determining the specific allele or the specific nucleotide carried by an individual at a polymorphic marker.

The present invention is suitable for identifying a bovine, including a young or adult bovine animal, an embryo, a semen sample, an egg, a fertilized egg, or a zygote, or other cell or tissue sample therefrom, to determine whether said bovine possesses the desired genotypes of the present invention, some of which are indicative of improved fertilization rate and embryonic survival.

Further provided is a method for genotyping the bovine STAT3 gene, comprising determining for the two copies of the STAT3 gene present the identity of the nucleotide pair at positions 25402 and 19069.

One embodiment of a genotyping method of the invention involves examining both copies of the STAT3 gene, or a fragment thereof, to identify the nucleotide pair at the polymorphic site in the two copies to assign a genotype to the individual. In some embodiments, “examining a gene” may include examining one or more of: DNA containing the gene, mRNA transcripts thereof, or cDNA copies thereof. As will be readily understood by the skilled artisan, the two “copies” of a gene, mRNA or cDNA, or fragment thereof in an individual may be the same allele or may be different alleles. In another embodiment, a genotyping method of the invention comprises determining the identity of the nucleotide pair at the polymorphic site.

The present invention further provides a kit for genotyping a bovine sample, the kit comprising in a container a nucleic acid molecule, as described above, designed for detecting the polymorphism, and optionally at least another component for carrying out such detection. Preferably, a kit comprises at least two oligonucleotides packaged in the same or separate containers. The kit may also contain other components such as hybridization buffer (where the oligonucleotides are to be used as a probe) packaged in a separate container. Alternatively, where the oligonucleotides are to be used to amplify a target region, the kit may contain, preferably packaged in separate containers, a polymerase and a reaction buffer optimized for primer extension mediated by the polymerase, such as PCR.

In one embodiment the present invention provides a breeding method whereby genotyping as described above is conducted on bovine embryos, and based on the results, certain cattle are either selected or dropped out of the breeding program.

Through use of the linked marker loci, procedures termed “marker assisted selection” (MAS) may be used for genetic improvement within a breeding nucleus; or “marker assisted introgression” for transferring useful alleles from a resource population to a breeding nucleus (Soller 1990; Soller 1994).

In previous studies, the present inventor demonstrated the ability of the IVF system to identify associations between candidate genes and fertility traits (Khatib et al., 2008a, b; Khatib et al., 2009). Here, the association was investigated of single gene and SNP-SNP interactions of STAT1 and STAT3 polymorphisms with fertilization rate and early embryonic survival using the IVF system. Truchet et al. (2004) hypothesized that because of the roles of STAT1 and STAT3 in the control of cell cycle and apoptosis, these two genes might have important roles in the early embryonic developmental stages. Indeed, the results disclosed herein show that single gene analyses revealed significant associations of STAT3 SNP25402 and STAT1 SNP213 with fertilization rate. Also, STAT3 SNP19069 was found to be associated with early embryonic survival. Moderate association was found of SNP19069 with fertilization and survival rates and of STAT1 SNP213 with fertilization rate.

Given that STAT1 and STAT3 proteins interact biologically by forming a heterodimer and translocating from the cytoplasm to the nucleus (Kodama et al., 1997), the present inventor also investigated the effects of the SNP-SNP interactions of these genes on fertility traits. The results showed highly significant association of STAT3 SNPs (SNP19609/SNP25402; P=3.690E-06) and of STAT1/STAT3 SNPs (STAT1/SNP19069; P=0.422E-06) with early embryonic survival. Given that single SNP analysis of STAT1 and STAT3 SNP25402 did not reveal highly significant associations with survival rate, these results testify to the merit of including epistsatic interactions in association studies (Carlborg and Haley, 2004).

For the significant interactions associated with survival rate, the observed survival rate for genotype combinations was calculated. For STAT3 SNP, the survival rates of embryos produced from AA/GG and that of CC/AA ovaries was extremely different than all other genotype combination. Likewise, for STAT1/SNP19069 the survival rate of embryos produced from TT/AA ovaries was relatively high compared to other genotype combinations. One can argue that the number of embryos carrying these genotypes is relatively small (Tables 3 and 4). Nevertheless for STAT3 SNPs, the difference between the second highest genotype combination (AC/AA) and the second lowest (AC/AG) observed survival rate was 8.7% (Table 3). For STAT1 SNP213/SNP19069 interaction, the difference between the second highest (TT/AG) and the lowest (TT/GG) observed survival rate for genotype combinations was 12.6% (Table 4). These differences in survival rate could not be observed in the single SNP analysis.

In a previous study, we reported that some genes in the POU1F1 pathway did not show significant associations with fertility traits using single gene analysis whereas gene-gene interactions were significant (Khatib et al., 2009). Thus, genotype combinations found to be associated with high embryonic survival rate in this study could be employed in gene-assisted selection programs aimed at improving fertility performance in dairy cattle.

STAT1 and STAT3 are members of the JAK/STAT signaling pathway. Truchet et al. (2004) reported that that STAT1 and STAT3 are present in mouse oocytes and in preimplantation embryos and that JAK/STAT pathway is functional during early embryonic development. Furthermore, in previous studies, we reported the several members of the JAK/STAT pathway were found to be associated with early embryonic survival in cattle. Taken together, our results provide additional support for the genetic involvement of JAK/STAT pathway in the early survival of cattle embryos.

In another embodiment, the present invention provides novel cattle genotyping, selective cattle breeding and related methods, based on the discovery that the above described STAT3 polymorphisms, in combination with a certain polymorphic form of the STAT1 gene, confers highly desirable fertilization and embryonic survival rate to cattle. The STAT1 polymorph, hereinafter referred to STAT1 SNP213, is depicted in FIG. 2, and was previously disclosed in U.S. patent application Ser. No. 11/179,581, the entire content of which is incorporated herein by reference. All the above description with regard to the SNPs in the STAT3 gene, including the terminology, the techniques and method of using, are equally applicable to STAT1 and the STAT1 SNP213.

The following examples are intended to illustrate preferred embodiments of the invention and should not be interpreted to limit the scope of the invention as defined in the claims.

EXAMPLES Materials and Methods

In-Vitro Fertilization and Embryo Production

Ovaries from mature Holstein cows (n=512) were collected from a local abattoir over a 3 yr period and immediately used in the IVF experiments. No genetic relationship information was available for these cows. Oocytes were aspirated from antral follicles and immediately incubated in maturation medium. On average, about 16.8 oocytes were aspirated from each ovary. Ovaries (n=67) from which fewer than 4 oocytes were harvested were excluded from the analysis. All oocytes aspirated from an ovary were combined with semen from one bull for an incubation period of 18-25 h as described in Khatib et al. (2008a,b). Semen of unrelated bulls (n=12) was purchased from different artificial insemination companies in Wisconsin. Fertilization rate was calculated as the number of cleaved embryos at 2 d post-fertilization out of the total number of oocytes exposed to sperm. After the fertilization period (fertilization=day 0), putative zygotes were stripped of their cumulus cells by vortexing and cultured until d 7. The first morphological evaluation of embryos was done on d 5 of culture. On d 5, embryos were evaluated for evidence of compaction or cell coalescence. Embryos undergoing compaction at this point will typically have 32-64 cells. On d 7 of culture, embryos previously classified as morulas (compacted) were re-evaluated for the presence of a blastocoele, whose presence classifies an embryo as a blastocyst. Survival rate of embryos was calculated as the number of normally developed blastocysts on d 7 out of the total number of embryos cultured. Standard error mean (SEM) of fertilization or survival rate was calculated by √{square root over ({circumflex over (p)}(1−{circumflex over (p)})/n)} where n was sample size and {circumflex over (p)} was fertilization or survival rate. A total of 7,519 oocytes were exposed to sperm, and a total of 5,075 embryos were produced.

Genotyping

DNA was extracted from ovaries (n=445) and semen samples (n=12) using standard phenol/chloroform protocols. The DNA concentrations were measured using a spectrophotometer (Ultraspec 2100; Amersham Biosciences). Three DNA pools were constructed from 50 different ovary samples to contain 50 ng of DNA from each sample and amplified with different sets of primers designed from the STAT3 gene, which is located on chromosome 19.

Amplification and SNP identification were as described in Khatib et al. (2008a,b). An A/G SNP was identified in exon 12 at position 19069 and an A/C SNP was identified in intron 19 at position 25402 (GenBank accession no. NC_(—)007314; region 43752931 to 43784155). For genotyping SNP19069, the primers exon12F (5′-TTCTACTTGAGCATGTACAGGG-3′) (SEQ ID NO: 3) and exon12R (5′-CTCTCCTGCTCAGCTATCATC 3)) (SEQ ID NO: 4) were used to amplify a 589-bp fragment.

The PCR products were digested with the restriction enzyme MSPA1I at 37° C. then electrophoresed on a 2.0% agarose gel. The A allele (cut) was indicated by the 384- and 205-bp fragments, and the G allele (uncut) was indicated by a single 589-bp fragment. For genotyping SNP25402, the primers intron19F (5′-AACTATGTTACTTGTGGCCC 3′) (SEQ ID NO: 5) and intron19R (5′-AACAGATCACCAGGCTCTCC-3′) (SEQ ID NO: 6) were used to amplify a 514-bp fragment. The PCR products were digested with the restriction enzyme HinfI which allows one to distinguish allele A (514-bp fragment) and allele C (167-bp and 347-bp fragments) when electrophoresed on a 2.0% agarose gel. STAT1 SNP (on chromosome 29) discovery and genotyping was as described in Cobanoglu et al. (2006). Allele frequencies of SNP were calculated by counting numbers of respective alleles in the study sample. Hardy-Weinberg equilibrium (HWE) was tested by Chi-square test.

Statistical Analysis

To test associations between ovary genotypes for the STAT1 and STAT3 SNPs and fertilization rate of oocytes or survival rate of embryos, a generalized linear model for binary response was fitted using the ‘logit’ link function (McCullagh and Nelder, 1989). The linear combination of predictors can be written as η₁ =b ₀+bull+STAT5A+Ovary Genotype where b₀ is a constant term, bull is the effect of the semen used to fertilize the oocytes, STAT5A is the effect of the ovary genotype of a STAT5A SNP that was found to be associated with fertilization rate and embryonic survival rate in a previous study (Khatib et al., 2008a), and Ovary Genotype is the effect of the ovary genotype of the SNP being tested. The response variable is coded as ‘0’ for unsuccessful fertilization/degenerate embryo or ‘1’ for successful fertilization/normal embryo. These two traits were modeled for each of the three SNPs (STAT1 SNP, STAT3 SNP25402 and STAT3 SNP19069) individually. Associations were tested using a Likelihood Ratio Test (LRT) by comparing the above model to a reduced model missing the Ovary Genotype predictor, which tested whether ovary genotype has an effect on fertilization success of oocytes or survival of embryos. LRT statistic is approximately distributed as χ_(v) ² with v degrees of freedom that is equal to the difference in numbers of parameters of the two nested models compared. When sample size is large, this approximation is quite accurate (McCullagh and Nelder, 1989). 95% confidence intervals for odds ratios between genotypes were calculated based on estimates and standard errors of the Genotype term in fitted models. The most frequent homozygotes were set as reference and had odds ratios of one. In addition, because of their biological interactions, 2-way interactions between the SNPs were also tested as described previously (Khatib et al., 2009). Briefly, a model including both SNPs and their interaction η₂ =b ₀+bull+STAT5A+Genotype1+Genotype2+Genotype1:Genotype2 was compared with a reduced model missing the Genotype1: Genotype2 term by LRT. This test was performed for each of the three interactions STAT1:SNP25402, STAT1:SNP19069 and SNP25402:SNP19069. Significant 2-way interactions indicate that the genotype effect of one SNP depends on the genotype of the interacting SNP. Therefore, fertilization or embryonic survival rates were calculated for each of the 9 genotype combinations for statistically significant interactions. Statistical analysis was performed using ‘glm’ function in R(R Development Core Team, 2008) version 2.8.1 and statistical significance was claimed at the 0.01 level.

Results

In this study we investigated the effects of three SNPs in the STAT1 and STAT3 genes and their interactions on fertility traits in Holstein cattle. In order to mimic pregnancy evaluation in live cows, we have constructed an IVF system which enables us to evaluate fertilization success and early embryonic survival, 2 initial components of pregnancy. We show that interactions between SNPs in the STAT3 gene and interactions between the STAT1 and STAT3 genes contribute significantly to the phenotypic variation in embryonic survival in cattle.

TABLE 1 Minor allele frequency (MAF) and Hardy-Weinberg-Equilibrium (HWE) test of SNPs Gene/SNP Alleles MAF HWE (p value) STAT3/SNP19069 A/G 0.475 0.566 STAT3/SNP25402 A/C 0.423 2.04E−07 STAT1 C/T 0.298 0.410

Table 1 shows minor allele frequencies and tests for HWE of the three SNPs. Only STAT3/SNP25402 showed strong evidence of disequilibrium. Table 2 shows the number of oocytes, fertilization rate, number of embryos and survival rate for the genotypic classes of STAT1 and STAT3 SNP. Single SNP analysis revealed statistically significant association (P=2.502E-05) between SNP25402 in STAT3 and fertilization rate. Oocytes collected from genotype AA ovaries showed 70.1% fertilization rate vs. 66.6% (odds ratio (OR)=0.83 as compared to AA) and 66.3% (OR=0.70) for oocytes collected from AC and CC ovaries, respectively (Table 2). Genotypes of SNP19069 in STAT3 and the STAT1 SNP showed moderate differences in fertilization rates (P=0.016 and P=0.014 respectively). For embryonic survival rate, only SNP19069 showed slight difference between STAT3 genotypes (P=0.010).

In order to quantify the contribution of the interaction between SNP of STAT3 and STAT1, a reduced model with bull and genotype information was compared with a full model including the interactions between SNP (Khatib et al., 2009). The interaction between STAT3 SNP (SNP19069/SNP25402) was highly significant for survival rate (P=3.690E-06) but not for fertilization rate. Also, the interaction between STAT1 SNP and SNP19069 was highly significant for survival rate (P=0.422E-06).

TABLE 2 Number of oocytes, fertilization rate (±SEM), number of embryos, survival rate (±SEM), and odds ratios for genotypic classes of STAT1 and STAT3 Genotype Odds Odds (No. of No. of Fertilization Ratio¹ No. of Survival Ratio Gene/SNP ovaries) oocytes rate ± SEM (95% CI) embryos rate ± SEM (95% CI) STAT3/ AA (119) 2,119 0.683 ± 0.010 1 1,448 0.353 ± 0.012 1 SNP19069 AG (229) 3,734 0.674 ± 0.008 [0.82, 1.05] 2,518 0.328 ± 0.009 [0.72, 0.96] GG (97) 1,641 0.662 ± 0.012 [0.68, 0.93] 1,086 0.330 ± 0.014 [0.63, 0.92] STAT3/ AA (169) 2,772 0.701 ± 0.009 1 1,942 0.329 ± 0.011 1 SNP25402 AC (155) 2,697 0.666 ± 0.009 [0.74, 0.94] 1,795 0.327 ± 0.011 [0.84, 1.13] CC (103) 1,852 0.663 ± 0.011 [0.60, 0.82] 1,228 0.365 ± 0.014 [0.88, 1.26] STAT1 CC (214) 3,538 0.688 ± 0.008 1 2,435 0.331 ± 0.009 1 CT (193) 3,441 0.663 ± 0.008 [0.78, 0.96] 2,282 0.334 ± 0.010 [0.85, 1.10] TT (35) 540 0.663 ± 0.020 [0.70, 1.04] 358 0.380 ± 0.026 [0.92, 1.48] ¹Odds ratios of the most frequent homozygotes were set to one as a reference

For significant interactions, we calculated the observed embryonic survival rates for each genotype combination of the 2-way interactions. As expected for a statistically significant interaction, genotypic effects of one SNP were dependent on the genotype of the interacting SNP. For example, for SNP25402/SNP19069 interaction, the survival rate of embryos produced from ovaries with AA genotype for SNP19069 (0.399) was markedly higher than that of embryos produced from GG ovaries (0.312), only when the genotype of SNP25402 was AC (Table 3). Embryos produced from AA/GG oocytes showed an extremely reduced survival rate (0.067) compared to all other genotypic combinations. However, it is worth noting that only 75 embryos carry the AA/GG combination. For the STAT1/SNP19069 interaction, the observed survival rate was 0.268 for embryos produced from TT/GG ovaries and 0.394 for embryos produced from TT/AG ovaries, yet this difference between GG and AG ovaries was reversed when STAT1 genotype was CC (0.368 vs 0.319). Taken together, these results strongly suggest a clear genetic interaction (epistasis) between genes that interact biologically.

TABLE 3 Observed survival rates (±SEM), numbers of embryos and average numbers of embryos per ovary for genotype combinations of SNP25402 and SNP19069 SNP25402/ SNP19069 AA (n¹, n²) AG GG AA 0.338 ± 0.014 0.343 ± 0.017 0.067 ± 0.029 (1097, 12.0)   (747, 11.5)  (75, 6.8) AC 0.339 ± 0.033 0.312 ± 0.012 0.325 ± 0.034  (218, 14.5) (1367, 11.1)   (188, 11.8) CC 0.627 ± 0.063 0.346 ± 0.026 0.359 ± 0.017   (59, 14.8)  (338, 11.6)  (786, 11.9) n¹= Number of embryos n²= Average number of embryos per ovary

TABLE 4 Observed survival rates (±SEM), numbers of embryos and average numbers of embryos per ovary for genotype combinations of STAT1 SNP and SNP19069 STAT1/ SNP19069 AA (n¹, n²) AG GG CC 0.327 ± 0.019 0.319 ± 0.013 0.368 ± 0.020  (587, 11.1) (1261, 11.1)   (549, 12.5) CT 0.361 ± 0.018 0.328 ± 0.014 0.295 ± 0.022  (753, 13.4) (1037, 11.3)   (440, 10.7) TT 0.508 ± 0.063 0.394 ± 0.035 0.269 ± 0.045   (63, 12.6)  (198, 11.0)  (97, 8.1) ¹Number of embryos ²Average number of embryos per ovary

While the invention has been described in connection with one or more embodiments, it should be understood that the invention is not limited to those embodiments, and the description is intended to cover all alternatives, modifications, and equivalents, as may be included within the spirit and scope of the appended claims.

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What is claimed is:
 1. A method for selectively breeding cattle, the method comprising superovulating a female bovine animal, collecting eggs from said superovulated female, in vitro fertilizing said eggs using sperm from a suitable male animal, developing said fertilized eggs into embryos, determining the identity of a nucleotide of a STAT3 gene of the embryo at a position corresponding to position 7342 of SEQ ID NO: 1, wherein the STAT3 gene comprises a nucleotide sequence of SEQ ID NO: 1, and implanting into a suitable female bovine animal a developing embryo that comprises the STAT3 gene of which does not homozygously have A at position 7342 of SEQ ID NO:
 1. 2. The method according to claim 1, further comprising determining the identity of a nucleotide of the bovine STAT1 gene corresponding to position 213 of SEQ ID NO: 2, wherein the STAT1 gene comprises a nucleotide sequence of SEQ ID NO: 2 and wherein the implanted embryo does not homozgously have C at position 213 of SEQ ID NO:
 2. 3. The method according to claim 1, further comprising determining the identity of a nucleotide of the bovine STAT3 gene of the embryo at a position corresponding to position 1009 of SEQ ID NO: 1, wherein the implanted embryo does not homozygously have G at position 1009 of SEQ ID NO:
 1. 4. The method according to claim 1, further comprising determining the identity of a nucleotide of the bovine STAT3 gene at a position corresponding to position 1009 of SEQ ID NO: 1 and determining the identity of a nucleotide of the bovine STAT1 gene at a position corresponding to position 213 of SEQ ID NO 2, and wherein the implanted embryo does not homozygously have G at position 1009 of SEQ ID NO: 1 and does not homogzyously have C at position 213 of SEQ ID NO:
 2. 5. The method according to claim 1, wherein the identity of the nucleotide is determined by obtaining a nucleic acid sample from the embryo.
 6. The method according, to claim 5, wherein the identity of the nucleotide is determined by sequencing.
 7. The method according to claim 2, wherein the identity of the nucleotide is determined by obtaining a nucleic acid sample from the embryo.
 8. The method according to claim 7, wherein the identity of the nucleotide is determined by sequencing.
 9. The method according to claim 3, wherein the identity of the nucleotide is determined by obtaining a nucleic acid sample from the embryo.
 10. The method according to claim 9, wherein the identity of the nucleotide is determined by sequencing.
 11. The method according to claim 5, wherein the identity of the nucleotide is determined by obtaining a nucleic acid sample from the embryo.
 12. The method according to claim 11, wherein the identity of the nucleotide is determined by sequencing. 